Our animal studies have demonstrated the therapeutic efficacy of antiinflammatory gene transfer in an animal model of rheumatoid arthritis (Makarov et al, 1995b), and revealed the problems to be solved on the way to clinical trials. The most crucial issue of gene therapy for arthritis concerns the choice of targets for intervention. As the inflammation is perpetuated by numerous proinflammatory molecules, the successful therapeutic approach should address the multiplicity of inflammatory pathways. Another important problem concerns the regulation of transgene expression. The long-term expression of the transgene should be regulated in such a manner that the transgene expression. The long-term expression of the transgene should be regulated in such a manner that the level of therapeutic gene products corresponds to the recurrent course of inflammation. To assess the prospects for long-term therapeutic treatment, it is obligatory to understand the mechanisms underlying the limited life span of the transgene. The first purpose of our proposed study is to assess the potential for two new targets for gene therapy of arthritis. 1. We will explore the feasibility of suppressing inflammation by targeting intracellular transcription factors. The profile of inflammatory molecules suggests the pivotal role for NF-kB in the perpetuation of inflammation in arthritis. Importantly, the efficacy of several remedies for arthritis correlates with their ability to inhibit NF-kB. Our studies have shown that NF-kB is activated in t he arthritic synovium, and NF-kB- mediated transcription is essential for the survival of synoviocytes in vitro. Thus activation of NF-kB in the synovium appears to be the point where the action of multiple inflammatory stimuli converges. It is our hypothesis the suppression of NF-kB in the synovium will provide a highly effective treatment for arthritis. We will test this hypothesis by using gene transfer of specific inhibitors of NF-kB. 2. Infiltrating leukocytes are the major source of proinflammatory mediators in arthritis. Gene transfer of inhibitory peptides will be attempted to block cell adherence and recruitment of leukocytes to inflamed joint. These peptides will be expressed in the synovium as a multimeric prodrug. Digestion of the prodrug by activated matrix proteinases will release active peptides providing inhibition of inflammation. Another proposed approach for the regulation of therapeutic gene products is based on developing vectors allowing for the inflammation-regulated expression of the transgene. NF- kB-inducible promoter/enhancer elements will serve as a prototype to evaluate the feasibility of this approach. Another purpose of this study is to examine the impact of apoptosis in synovial cells on the duration of the transgene expression. Our studies have shown that apoptosis is a common feature of synovial cells in normal as well as in inflamed joints. Thus it is one goal of this proposal to examine apoptosis in transduced cells as a fundamental factor limiting the persistence of the transgene in the transgene in the synovium. Successful implementation of the proposed project should provide significant progress towards developing clinically relevant approaches for the gene therapy of arthritis.